Communication with the environment is essential for survival and proper functionality of individual cells within an organism. One element of such communication is exchange of components between the intracellular and extracellular space. It includes two major processes, secretion and endocytosis, which are roughly equivalent to export and import, respectively, in the human world. This project will focus on the mechanisms of the major endocytic pathway, clathrin-mediated endocytosis (CME), by which cells take up exogenous molecules and cell surface components in a highly selective way. The key step of CME is the initial formation of an endocytic vesicle. This process consists of: (i) Assembly of the clathrin-based coat, a multiprotein scaffold recruiting the cargo and the endocytic machinery to the sites of endocytosis~ (ii) invagination of the coated plasma membrane to form a clathrin-coated pit~ (iii) elongation of the pit and constrictions of its neck t form a clathrin-coated bud~ (iv) scission of the bud neck to form an endocytic vesicle~ and (v) inward movement of the vesicle. All these processes are energetically unfavorable and require force-generating machinery to occur. The ongoing research on the mechanisms of CME increasingly points to the actin cytoskeleton as an important component of the molecular machinery driving endocytic vesicle internalization. However, an explicit model for the specific roles of actin cytoskeleton in CME has not been formulated because of a lack of high resolution structural information about the cytoskeletal architecture at endocytic sites. The major reasons for this deficiency are an extremely small size and transient nature of actin patches associated with the endocytic sites, dense packing of individual actin filaments within these patche making them irresolvable by light microscopy, and a well-known difficulty of preserving dynamic actin filament networks for electron microscopy. Using our special expertise in platinum replica electron microscopy that is most useful for the analysis of the cytoskeletal architecture, we propose to determine the structural organization and molecular composition of actin filament arrays associated with various types of clathrin-coated structures, to correlate the changes in the cytoskeleton organization with different stages of formation and maturation of clathrin-coated structures, and establish roles of several key proteins in this process by functional approaches. By these studies, we will test a hypothesis that a branched actin network nucleated around the perimeter of a clathrin-coated pit exerts pushing force onto all three surfaces: the growing bud, the bud neck, and the plasma membrane at the base of a bud, in order to constrict and elongate the bud neck, but then it is rearranged into a comet tail that propels the newly formed vesicle into the cytoplasm. The results of these studies will significantly advance our understanding of the actin-dependent mechanisms of vesicle internalization during CME.